MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF ANAMORPHIC FUNGI Hugo Madrid Lorca Dipòsit Legal: T. 1355-2011 ADVERTIMENT. La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX (www.tesisenxarxa.net) ha estat autoritzada pels titulars dels drets de propietat intel·lectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposició des d’un lloc aliè al servei TDX. No s’autoritza la presentació del seu contingut en una finestra o marc aliè a TDX (framing). Aquesta reserva de drets afecta tant al resum de presentació de la tesi com als seus continguts. En la utilització o cita de parts de la tesi és obligat indicar el nom de la persona autora. ADVERTENCIA. La consulta de esta tesis queda condicionada a la aceptación de las siguientes condiciones de uso: La difusión de esta tesis por medio del servicio TDR (www.tesisenred.net) ha sido autorizada por los titulares de los derechos de propiedad intelectual únicamente para usos privados enmarcados en actividades de investigación y docencia. No se autoriza su reproducción con finalidades de lucro ni su difusión y puesta a disposición desde un sitio ajeno al servicio TDR. No se autoriza la presentación de su contenido en una ventana o marco ajeno a TDR (framing). Esta reserva de derechos afecta tanto al resumen de presentación de la tesis como a sus contenidos. En la utilización o cita de partes de la tesis es obligado indicar el nombre de la persona autora. WARNING. On having consulted this thesis you’re accepting the following use conditions: Spreading this thesis by the TDX (www.tesisenxarxa.net) service has been authorized by the titular of the intellectual property rights only for private uses placed in investigation and teaching activities. Reproduction with lucrative aims is not authorized neither its spreading and availability from a site foreign to the TDX service. Introducing its content in a window or frame foreign to the TDX service is not authorized (framing). This rights affect to the presentation summary of the thesis as well as to its contents. In the using or citation of parts of the thesis it’s obliged to indicate the name of the author. UNIVERSITAT ROVIRA I VIRGILI MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF ANAMORPHIC FUNGI Hugo Madrid Lorca DL: T. 1355-2011 a Molecular and Phenotypic Characterization of Anamorphic Fungi Hugo Madrid Lorca Doctoral Thesis Directed by Drs. Josep Guarro Artigas, Josep Cano Lira and Josepa Gené Díaz Facultat de Medicina i Ciències de la Salut Departament de Ciències Mèdiques Bàsiques Reus 2011 UNIVERSITAT ROVIRA I VIRGILI MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF ANAMORPHIC FUNGI Hugo Madrid Lorca DL: T. 1355-2011 UNIVERSITAT ROVIRA I VIRGILI MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF ANAMORPHIC FUNGI Hugo Madrid Lorca DL: T. 1355-2011 UNIVERSITAT ROVIRA I VIRGILI MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF ANAMORPHIC FUNGI Hugo Madrid Lorca DL: T. 1355-2011 UNIVERSITAT ROVIRA I VIRGILI MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF ANAMORPHIC FUNGI Hugo Madrid Lorca DL: T. 1355-2011 To those who live and breathe mycology UNIVERSITAT ROVIRA I VIRGILI MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF ANAMORPHIC FUNGI Hugo Madrid Lorca DL: T. 1355-2011 UNIVERSITAT ROVIRA I VIRGILI MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF ANAMORPHIC FUNGI Hugo Madrid Lorca DL: T. 1355-2011 Acknowledgments I would like to show my gratitude to Drs. Josep Guarro, Josepa Gené, Josep Cano and Alberto Stchigel for giving me the opportunity to work with them. I feel lucky to have worked in such a prestigious center. Thanks for the mycological lessons and for giving me the chance to work in fungal biodiversity! I am heartily thankful to Drs. Victor Silva and Eduardo Piontelli for guidance and support years ago, when I started working in microbiology. Your enthusiasm breathes life into Chilean mycology! I am grateful to Dr. Sybren de Hoog (CBS; Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) for accepting me in his research group for 3 months and for his patience, encouragement and sense of humor. I also want to express my gratitude to other people who helped me during my stay at the CBS: Manon Verweij, Tineke van den Berg-Visser, Bert Gerrits van den Ende, Kasper Luijsterburg, Mohammad Javad Najafzadeh, Stephanie Miesch, and Qiaoyun Lu. I am thankful to Dr. Estibaliz Mateo Alesanco for her continuous support during my stay in Utrecht. I appreciate the company and help of my colleagues at Universitat Rovira i Virgili: Eduardo Alvarez, Haybrig Perdomo, Margarita Hernandez, Fabiola Fernández, Valentina Salas and Carolina Silvera. Most of the micrographs used in the introduction of this thesis were kindly provided by M. Hernández and C. Silvera. I am also grateful to Cati Nuñez and Nuria Pilas for their technical assistance. I owe my deepest gratitude to the people who have given me true love, respect, support and true friendship during these years. Like nature, life is cyclic: all the good you do will come back to you. UNIVERSITAT ROVIRA I VIRGILI MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF ANAMORPHIC FUNGI Hugo Madrid Lorca DL: T. 1355-2011 UNIVERSITAT ROVIRA I VIRGILI MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF ANAMORPHIC FUNGI Hugo Madrid Lorca DL: T. 1355-2011 INDEX Page List of frequently used abbreviations………………………………………………….....i 1. INTRODUCTION………………………………………………………………..1 1.1 The kingdom Fungi: characteristics, ecology, and relevance to man………….1 1.2 The fungal life cycle…………………………………………………………...2 1.3 Fungal taxonomy and nomenclature…………………………………………...4 1.4 Taxonomy and morphology of anamorphic fungi……………………………10 1.5 DNA sequence analyses of anamorphic fungi………………………………..24 2. INTEREST OF THIS WORK AND OBJECTIVES……………………….....33 3. MATERIALS AND METHODS………………………………………………39 3.1 Origin of the isolates and methods for fungal isolation……………………....41 3.1.1 Clinical isolates of Sporothrix…….…………………...…………….....41 3.1.2 Isolation of soil-borne anamorphic fungi..………………...…………...41 3.2 Phenotypic characterization…………………………………………………..42 3.2.1 Morphology…………………………………………………………….42 3.2.2 Physiological tests………………………………………………………43 3.3 Molecular studies……………………………………………………………..43 4. RESULTS…………………………………………………………………………...47 4.1 Sporothrix brunneoviolacea and Sporothrix dimorphospora, two new members of the Ophiostoma stenoceras-Sporothrix schenckii complex…………49 4.2 Sporothrix globosa, a pathogenic fungus with widespread geographical distribution…………..…………………………………………………………....63 UNIVERSITAT ROVIRA I VIRGILI MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF ANAMORPHIC FUNGI Hugo Madrid Lorca DL: T. 1355-2011 4.3 Ramophialophora humicola and Fibulochlamys chilensis, two new microfungi from soil………….………………………………………….….........71 4.4 Two new species of Cladorrhinum…………………………………………..81 4.5 A new species of Leptodiscella from Spanish soil………………………….109 5. SUMMARIZING DISCUSSION…………………………………………………127 5.1 New members of the Ophiostoma stenoceras-Sporothrix schenckii complex and thermal dimorphism in Ophiostoma bragantinum………………………….129 5.2 Distribution of Sporothrix globosa and other pathogenic species of Sporothrix……………………………………………………………………….132 5.3 Three new Phialophora-like fungi: Cladorrhinum flexuosum, C. microsclerotigenes and Ramophialophora humicola…………………………...134 5.4 Fibulochlamys chilensis, a new basidiomycetous anamorph from Chilean soil………………………………………………………………………………139 5.5 Leptodiscella brevicatenata, a new anamorphic member of the Dothideomycetes with appendaged conidia…………………………………....141 6. CONCLUSIONS…………………………………………………………………..145 7. REFERENCES....…...……………………………………………………………149 UNIVERSITAT ROVIRA I VIRGILI MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF ANAMORPHIC FUNGI Hugo Madrid Lorca DL: T. 1355-2011 List of frequently used abbreviations ATCC American Type Culture Collection, Bethesda, USA BAFC Universidad de Buenos Aires Herbarium BAYER Bayer Healthcare, Wupertal, Germany BLAST Basic Local Alignment Search Tool bp base pair(s) BPI Systematic Botany and Mycology Laboratory, Beltsville, USA BRIP Queensland Department of Primary Industries Plant Pathology Herbarium, Brisbane, Australia BT2 a fragment of the β-tubulin gene Buck William Buck, Jacksonville, Florida ca. circa, Latin word meaning "around","about" or "approximately" CAL calmodulin gene CBS Centraalbureau voor Schimmelcultures, Utrecht, Netherlands CBSC Carolina Biological Supply Company, Burlington, USA CI consistency index CMA corn meal agar CMW culture collection of the Forestry and Agricultural Biotechnology Institute, Pretoria, South Africa comb. nov. combinatio nova, Latin expression meaning "new combination" d day(s) D1/D2 D1/D2 variable domain of the 28S rRNA gene DAOM National Mycological Herbarium, Ottawa, Canada DNA deoxyribonucleic acid DRBC dichloran rose Bengal chloramphenicol agar e.g. exempli gratia, Latin expression meaning "for example" i UNIVERSITAT ROVIRA I VIRGILI MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF ANAMORPHIC FUNGI Hugo Madrid Lorca DL: T. 1355-2011 List of frequently used abbreviations EMBL European Molecular Biology Laboratory et al. et alii (Latin expression meaning "and others") etc. et cetera, Latin expression meaning "and the rest (of such things)" F Field Museum of Natural History, Chicago, USA fam. nov. familia nova, Latin expression meaning “new family” Fig. figure FMR Faculty of Medicine culture collection, Universitat Rovira i Virgili,